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University of Cincinnati College of Medicine

Probenecid was initially developed with the goal of reducing the renal excretion of antibiotics, specifically penicillin. It is still used for its uricosuric properties in the treatment in gout, but its clinical relevance has sharply fallen and is rarely used today for either. Interestingly, throughout the last 60 years, there have been a host of apparently unrelated studies using probenecid in the clinical and basic research arena, including its potential use in the diagnosis and treatment of depression and its use to prevent fura-2 leakage in calcium transient studies. Recently, it has been shown that it is also an agonist of the Transient Receptor Potential Vanilloid 2 channel. Due to its unique action and new findings implicating TRPV channels in physiology and in disease, probenecid may have a new future as a research tool, and perhaps as a clinical agent in the neurology and cardiology fields. We review the history of probenecid in this paper and its potential future uses.

The history and future of probenecid.

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation.

Ischemic heart disease is characterized by contractile dysfunction and increased cardiomyocyte death, induced by necrosis and apoptosis. Increased cell survival after an ischemic insult is critical and depends on several cellular pathways, which have not been fully elucidated.

Novel role of HAX-1 in ischemic injury protection involvement of heat shock protein 90.

The current therapeutic options for acute decompensated heart failure are limited to afterload reducers and positive inotropes. The latter increases myocardial contractility through changes in myocyte calcium (Ca²⁺) handling (mostly through stimulation of the β-adrenergic pathways [β-ADR]) and is associated with paradoxical effects of arrhythmias, cell death, and subsequently increased mortality. We have previously demonstrated that probenecid can increase cytosolic Ca²⁺ levels in the cardiomyocyte resulting in an improved inotropic response in vitro and in vivo without activating the β-ADR system. We hypothesize that, in contrast to other commonly used inotropes, probenecid functions through a system separate from that of β-ADR and hence will increase contractility and improve function without damaging the heart. Furthermore, our goal was to evaluate the effect of probenecid on cell death in vitro and its use in vivo as a positive inotrope in a mouse model of ischemic cardiomyopathy. Herein, we demonstrate that probenecid induced an influx of Ca²⁺ similar to isoproterenol, but does not induce cell death in vitro. Through a series of in vivo experiments we also demonstrate that probenecid can be used at various time points and with various methods of administration in vivo in mice with myocardial ischemia, resulting in improved contractility and no significant difference in infarct size. In conclusion, we provide novel data that probenecid, through its activity on cellular Ca²⁺ levels, induces an inotropic effect without causing or exacerbating injury. This discovery may be translatable if this mechanism is preserved in man.

Probenecid as a noninjurious positive inotrope in an ischemic heart disease murine model.

Cardiovascular disease is a leading cause of morbidity and mortality worldwide, encompassing a variety of cardiac and vascular conditions. Transient receptor potential vanilloid (TRPV) channels, specifically TRPV type 1 (TRPV1) and TRPV type 2 (TRPV2), are relatively recently described channels found throughout the body including within and around the cardiovascular system. They are activated by a variety of stimuli including high temperatures, stretch, and pharmacologic and endogenous ligands. The TRPV1 channel has been found to be an important player in the pathway of the detection of chest pain after myocardial injury. Activation of peripheral TRPV1 via painful stimuli or capsaicin has been shown to have cardioprotective effects, whereas genetic abrogation of TRPV1 results in increased myocardial damage after ischemia and reperfusion injury in comparison to wild-type mice. Furthermore, blood pressure changes have been noted upon TRPV1 stimulation. Similarly, the TRPV2 channel has also been associated with changes in blood pressure and cardiac function depending on how and where the channel is activated. Interestingly, overexpression of TRPV2 channels in the heart induces dystrophic cardiomyopathy; however, stimulation under physiologic conditions leads to improved cardiac function. Probenecid, a TRPV2 agonist, has been studied as a model therapy for its inotropic effects and potential use in the treatment of cardiomyopathy. In this review, we present an up to date account of the growing evidence that supports the study of TRPV1 and TRPV2 channels as targets for therapeutic agents of cardiovascular diseases.

Targeting TRPV1 and TRPV2 for potential therapeutic interventions in cardiovascular disease.

Detailed studies in animal models to assess the importance of aging animals in cardiovascular research are rather scarce. The increase in mouse models used to study cardiovascular disease makes the establishment of physiologic aging parameters in myocardial function in both male and female mice critical. Forty-four FVB/N mice were studied at multiple time points between the ages of 3 and 16 mo using high-frequency echocardiography. Our study found that there is an age-dependent decrease in several systolic and diastolic function parameters in male mice, but not in female mice. This study establishes the physiologic age- and gender-related changes in myocardial function that occur in mice and can be measured with echocardiography. We report baseline values for traditional echocardiography and advanced echocardiographic techniques to measure discrete changes in cardiac function in the commonly employed FVB/N strain.

Age- and gender-related changes in ventricular performance in wild-type FVB/N mice as evaluated by conventional and vector velocity echocardiography imaging: a retrospective study.

Valvular disease is common in patients with Marfan syndrome and can lead to cardiomyopathy. However, some patients develop cardiomyopathy in the absence of hemodynamically significant valve dysfunction, suggesting alternative mechanisms of disease progression. Disruption of LDL receptor-related protein-1 (Lrp1) in smooth muscle cells has been shown to cause vascular pathologies similar to Marfan syndrome, with activation of smooth muscle cells, vascular dysfunction and aortic aneurysms. This study used echocardiography and blood pressure monitoring in mouse models to determine whether inactivation of Lrp1 in vascular smooth muscle leads to cardiomyopathy, and if so, whether the mechanism is a consequence of valvular disease. Hemodynamic changes during treatment with captopril were also assessed. Dilation of aortic roots was observed in young Lrp1-knockout mice and progressed as they aged, whereas no significant aortic dilation was detected in wild type littermates. Diastolic blood pressure was lower and pulse pressure higher in Lrp1-knockout mice, which was normalized by treatment with captopril. Aortic dilation was followed by development of aortic insufficiency and subsequent dilated cardiomyopathy due to valvular disease. Thus, smooth muscle cell Lrp1 deficiency results in aortic dilation and insufficiency that causes secondary cardiomyopathy that can be improved by captopril. These findings provide novel insights into mechanisms of cardiomyopathy associated with vascular activation and offer a new model of valvular cardiomyopathy.

Smooth muscle LDL receptor-related protein-1 deletion induces aortic insufficiency and promotes vascular cardiomyopathy in mice.

Transient receptor potential cation channels have been implicated in the regulation of cardiovascular function, but only recently has our laboratory described the vanilloid-2 subtype (TRPV2) in the cardiomyocyte, though its exact mechanism of action has not yet been established. This study tests the hypothesis that TRPV2 plays an important role in regulating myocyte contractility under physiological conditions. Therefore, we measured cardiac and vascular function in wild-type and TRPV2(-/-) mice in vitro and in vivo and found that TRPV2 deletion resulted in a decrease in basal systolic and diastolic function without affecting loading conditions or vascular tone. TRPV2 stimulation with probenecid, a relatively selective TRPV2 agonist, caused an increase in both inotropy and lusitropy in wild-type mice that was blunted in TRPV2(-/-) mice. We examined the mechanism of TRPV2 inotropy/lusitropy in isolated myocytes and found that it modulates Ca(2+) transients and sarcoplasmic reticulum Ca(2+) loading. We show that the activity of this channel is necessary for normal cardiac function and that there is increased contractility in response to agonism of TRPV2 with probenecid.

ArticleTotal : 2	Year
Publication title Cited by 16	2014
Publication title Cited by 54	2014

Article	Year
The history and future of probenecid. Cardiovascular toxicology\| PubMed ID: 21938493	2012
Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation. Journal of molecular and cellular cardiology\| PubMed ID: 22561103	2012
Novel role of HAX-1 in ischemic injury protection involvement of heat shock protein 90. Circulation research\| PubMed ID: 22982986	2013
Probenecid as a noninjurious positive inotrope in an ischemic heart disease murine model. Journal of cardiovascular pharmacology and therapeutics\| PubMed ID: 23241275	2013
Targeting TRPV1 and TRPV2 for potential therapeutic interventions in cardiovascular disease. Translational research : the journal of laboratory and clinical medicine\| PubMed ID: 23453732	2013
Age- and gender-related changes in ventricular performance in wild-type FVB/N mice as evaluated by conventional and vector velocity echocardiography imaging: a retrospective study. Ultrasound in medicine & biology\| PubMed ID: 23791351	2013
Smooth muscle LDL receptor-related protein-1 deletion induces aortic insufficiency and promotes vascular cardiomyopathy in mice. PloS one\| PubMed ID: 24312398	2013
Novel role of transient receptor potential vanilloid 2 in the regulation of cardiac performance. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 24322617	2014

Nathan Robbins

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